Longitudinal assay

ABSTRACT

Embodiments of the invention include methods for detecting and determining the concentrations of macro and small molecules, including bio-molecules, in a liquid or gaseous sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of co-pending International PatentApplication No. PCT/US10/58112, filed 24 Nov. 2010, which is herebyincorporated herein.

BACKGROUND OF THE INVENTION

Many areas of research and clinical practice employ various detectionmethods and technologies for detecting and measuring concentrations ofmacro and small molecules, including bio-molecules. Often, it isdesirable to perform such detection with great sensitivity, quickly,and/or in a multiplex. Known methods, however, typically sacrifice oneor more features in favor of another, depending on the context in whichthe method is employed.

For example, multiplexed assays typically involve detection of allanalytes after a single period of elapsed time. This period is oftenchosen based on the optimal binding kinetics of one analyte of aplurality of analytes, which necessarily results in detection at a timethat is not optimal for the other analytes, based on their bindingkinetics. Similarly, known methods may sacrifice the sensitivity of anassay in favor of obtaining a rapid result, or vice versa.

A significant limitation of known assay methods is their inability todistinguish specific binding of an analyte from non-specific binding ofnon-analyte components of a tested sample. Biological samples, forexample, may contain a high concentration of proteins that are not ofinterest, relative to the concentration of a protein that is ofinterest, but which may still engage in non-specific binding with adetection label or with a capture agent or other moiety. A singlemeasure of binding (e.g., based on fluorescence of the detection label)will therefore necessarily include both specific binding andnon-specific binding components and result in overestimation of thedegree of specific binding. In some cases, a signal may be present dueto non-specific interactions alone without any specific component.However, a single measure of binding will not be able to distinguishbetween a true (specific) signal, a false (non-specific) signal, or amix of both.

Furthermore, non-specific binding can often be relatively stable overtime. This leads to significant complications and limitations forapplications such as biomarker discovery and validation, whereinnon-specific signals can taint results and lead to the expensive andlengthy pursuit of biomarkers that are ultimately determined to be of novalue or significance. Additionally, in clinical diagnostic settings,non-specific data leads to higher false positive rates, and limits theability to accurately quantify biomarker concentrations.

BRIEF DESCRIPTION OF THE INVENTION

Embodiments of the invention relate generally to macro and smallmolecule detection and, more particularly, to methods for detectingmacro and small molecules, including bio-molecules, in a liquid orgaseous sample. Methods according to embodiments of the invention areuseful in the identification, discovery, and validation of biomarkers,as well as the screening of individuals for such biomarkers fordiagnostic, therapeutic, and forensic purposes.

In one embodiment, the invention provides a method of detecting ananalyte in a fluid sample, the method comprising: passing a fluid samplecontaining a labeled analyte across at least one assay surfacecontaining a capture agent for the analyte; detecting the labeledanalyte; repeating the passing and detecting steps at least once; andcreating a binding curve for the analyte based on the detecting of thelabeled analyte. As used herein, an “analyte” refers to a moleculespecifically targeted by the capture agent in a given assay, e.g. anantigen (analyte) being targeted by its antibody pair (capture agent),or vice versa.

In another embodiment, the invention provides a method of detecting ananalyte in a fluid sample, the method comprising: passing a fluid samplecontaining a labeled analyte across at least one assay surfacecontaining a capture agent for the analyte; passing a non-labeledsolution across the at least one assay surface; detecting the labeledanalyte; repeating the passing steps and the detecting step at leastonce; and creating a binding curve for the analyte based on thedetecting of the labeled analyte.

In still another embodiment, the invention provides a method ofdetecting an analyte in a fluid sample, the method comprising: passing afluid sample containing an analyte across at least one assay surfacecontaining a capture agent for the analyte; passing a solutioncontaining a labeled molecule across the at least one assay surface, thelabeled molecule being capable of binding to the analyte; passing anon-labeled solution across the at least one assay surface; detectingthe labeled molecule; repeating the passing steps and the detecting atleast once; and creating a binding curve for the analyte based on thedetecting of the labeled molecule.

In yet another embodiment, the invention provides a method ofdistinguishing specific binding of an analyte and capture agent fromnon-specific binding of the analyte and/or the capture agent, the methodcomprising: obtaining a binding curve for a given capture agent and itsinteraction with the sample based on a plurality of temporally-spaceddetections of the capture agent to sample binding interactions within anassay (e.g. by measuring the intensity of an optical label directly orindirectly attached to the analyte and/or non-analytes within the samplebinding to said capture agent, or by measuring the binding of theanalyte and/or non-analytes using any known or later-developed method ortechnique); determining a first detection intensity of the capture agentto sample binding interactions and a first temporal point in the bindingcurve at which a rise to an equilibrium may be inferred; and attributingto non-specific binding a first portion of at least one detectedintensity subsequent to the first temporal point that is equal to thefirst detection intensity.

In still another embodiment, the invention provides a method ofdistinguishing specific binding of an analyte and capture agent fromnon-specific binding of the analyte and/or the capture agent, the methodcomprising: obtaining a binding curve for an analyte based on aplurality of temporally-spaced detections of the capture agent to samplebinding interactions within an assay (e.g. by measuring the intensity ofan optical label directly or indirectly attached to the analyte and/ornon-analytes within the sample binding to said capture agent, or bymeasuring the binding of the analyte and/or non-analytes using any knownor later-developed method or technique); determining a first detectionintensity of the capture agent to sample binding interaction and a firsttemporal point in the binding curve at which a rise to an equilibriumhas been reached; and attributing the first detection intensity tonon-specific binding.

In yet another embodiment, the invention provides a method ofidentifying biomarkers for therapeutic and/or diagnostic use, the methodcomprising: obtaining a binding curve for an analyte based on aplurality of temporally-spaced detections of the capture agent to samplebinding interactions within an assay (e.g. by measuring the intensity ofan optical label directly or indirectly attached to the analyte and/ornon-analytes within the sample binding to said capture agent, or bymeasuring the binding of the analyte and/or non-analytes using any knownor later-developed method or technique); determining a first detectionintensity of the capture agent to sample binding interactions and afirst temporal point in the binding curve at which a rise to anequilibrium may be inferred; attributing to non-specific binding a firstportion of at least one detected intensity subsequent to the firsttemporal point that is equal to the first detection intensity;attributing to specific binding of the analyte a second portion of atleast one detected intensity subsequent to the first temporal pointequal to a difference between the at least one detected intensity andthe first detection intensity; and determining an initial concentrationof the analyte based on the specific binding.

In still another embodiment, the invention provides a method ofmeasuring a biomarker concentration in a biological sample of anindividual, the method comprising: obtaining from an individual abiological sample; creating a binding curve for a biomarker within thebiological sample; determining a first detection intensity and a firsttemporal point in the binding curve at which a rise to an equilibriummay be inferred; attributing to non-specific binding of the captureagent a first portion of at least one detected intensity subsequent tothe first temporal point that is equal to the first detection intensity;attributing to specific binding of the capture agent to the biomarker asecond portion of at least one detected intensity subsequent to thefirst temporal point equal to a difference between the at least onedetected intensity and the first detection intensity; and determining aninitial concentration of the biomarker based on the specific binding ofthe biomarker.

In some embodiments, a method to distinguish specific binding fromnon-specific binding will be used to identify if a signal in a givenassay is the result of a specific binding event, a non-specific bindingevent, or both. In the event that the signal from a given assay is aresult of a specific binding event only, there may not be a firstdetection intensity and a first temporal point in the binding curve atwhich a rise to an equilibrium has been reached. In such cases, whereequilibrium has not been reached within a predefined period (e.g., 30minutes, 40 minutes, 50 minutes, 60 minutes, etc.) which may vary basedat least in part on the binding kinetics of the assay, and the signalrises at a linear or near linear rate through the assay, that signal maybe treated as a specific signal.

In the event that the assay signal is a result of both specific andnon-specific binding events, methods according to embodiments of theinvention may be used to isolate the specific component of the bindingcurve from the non-specific component, to improve quantitative analysisof the target analytes (e.g. calculation of initial target analyteconcentration).

In another embodiment, the invention provides a method of identifyingbiomarkers for therapeutic and/or diagnostic use, the method comprising:obtaining a binding curve for an analyte in a sample, based on aplurality of temporally-spaced detections of the capture agent to samplebinding interactions within an assay containing the sample (e.g. bymeasuring the intensity of an optical label directly or indirectlyattached to the analyte and/or non-analytes within the sample binding tosaid capture agent, or by measuring the binding of the analyte and/ornon-analytes using any known or later-developed method or technique);determining a first detection intensity and a first temporal point inthe binding curve at which a rise to an equilibrium has been reached;attributing to non-specific binding of at least one non-analyte a firstportion of at least one detected intensity subsequent to the firsttemporal point that is equal to the first detection intensity;attributing to specific binding of the analyte a second portion of atleast one detected intensity subsequent to the first temporal pointequal to a difference between the at least one detected intensity andthe first detection intensity; and determining an initial concentrationof the analyte in the sample based on the specific binding.

In still another embodiment, the invention provides a method ofmeasuring a biomarker concentration in a biological sample of anindividual, the method comprising: obtaining from an individual abiological sample; creating a binding curve for the biomarker, based ona plurality of temporally-spaced detections of the capture agent tosample binding interactions within an assay containing the biologicalsample (e.g. by measuring the intensity of an optical label directly orindirectly attached to the analyte and/or non-analytes within the samplebinding to said capture agent, or by measuring the binding of theanalyte and/or non-analytes using any known or later-developed method ortechnique); determining a first detection intensity and a first temporalpoint in the binding curve at which a rise to an equilibrium has beenreached; attributing to non-specific binding of small and/or macromolecules other than the biomarker a first portion of at least onedetected intensity subsequent to the first temporal point that is equalto the first detection intensity; attributing to specific binding of thebiomarker a second portion of at least one detected intensity subsequentto the first temporal point equal to a difference between the at leastone detected intensity and the first detection intensity; anddetermining an initial concentration of the biomarker based on thespecific binding.

In certain embodiments, an assay may refer to an array of captureagents, specific to target analytes, spotted onto a surface (through anymethod familiar to those skilled in the art such as using contact or noncontact printing devices) in known locations such that the resultantbinding interactions of analytes or non-analytes to the capture agent(s)can be detected and analyzed, and in the event an analyte is present, itcan be identified through reference to the known capture agent it bindsto.

In other embodiments, an assay may refer to an array of a biologicalsample such as a cell lysate, tissue samples, or fractions thereof, orother biological samples that may be of interest for biomarkerdiscovery, validation or screening; wherein the biological sample isspotted onto a surface at known locations, such that resultant bindinginteractions of analytes or non-analytes to capture agents within thebiological sample can be detected an analyzed, and in the even ananalyte of interest is present (e.g. a specific interaction is detectedwith a given fraction of a cell lysate spotted on a array surface), itcan be identified through further examination/analysis of the known spotof the biological sample; e.g. through further analysis of a givenfraction of a cell lysate, either through further iterativefractionation and arraying, and/or through mass spectrometry and/orthrough other methods known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of this invention will be more readilyunderstood from the following detailed description of the variousaspects of the invention taken in conjunction with the accompanyingdrawings that depict various embodiments of the invention, in which:

FIGS. 1A through 1C show schematic top and side cross-sectional views ofa cartridge device and its use according to various embodiments of theinvention.

FIGS. 2 and 3 show flow diagrams of various methods according toembodiments of the invention.

FIGS. 4A through 4C show schematic side cross-sectional views of acartridge device and its use according to various embodiments of theinvention.

FIGS. 5 and 6 show flow diagrams of various methods according toembodiments of the invention.

FIGS. 7 through 9 show schematic representations of binding curvescreated using methods according to various embodiments of the invention.

FIG. 10 shows a flow diagram of a method according to an embodiment ofthe invention.

It is noted that the drawings of the invention are not to scale. Thedrawings are intended to depict only typical aspects of the invention,and therefore should not be considered as limiting the scope of theinvention. In the drawings, like numbering represents like elementsamong the drawings.

DETAILED DESCRIPTION OF THE INVENTION

Turning now to the drawings, FIG. 1A shows a schematic top view of acartridge 100 according to an embodiment of the invention. Cartridge 100includes an assay surface 10 upon which are disposed a plurality ofcapture agent spots 20-28. Capture agents suitable for use inembodiments of the invention include, for example, antibodies (includingautoantibodies), antigens (including native antigens), proteins,peptides, complexes of antibodies and antigens, complexes of proteins,lipids, cell or tissue lysates and fractions thereof, DNA, RNA, andother molecular or elemental moieties or complexes thereof, capable ofbinding to and forming a complex with an analyte of interest, as will berecognized by one skilled in the art. Capture agent spots 20-28therefore comprise collections of capture agents adhered to assaysurface 10, such that corresponding analytes (e.g., a protein, apeptide, an antibody, an auto-antibody, a native antigen, a proteincomplex (including complexes of antibodies, antigens, native antigensand autoantibodies), a lipid, DNA, RNA, etc.), when passed acrosscapture agent spots 20-28 will tend to bind to the capture agents,thereby forming an analyte-capture agent complex amenable to detectionand measurement. Each capture agent spot 20-28 may include the samecapture agent or different capture agents. Similarly, while captureagent spots 20-28 are shown in FIG. 1A in a substantially straight line,other arrangements are also possible and in some cases desirable, aswill be described below.

Cartridge 100 includes an inlet 30 and outlet 34 through which aninfluent 32 and effluent 36, respectively, pass, thereby permitting flow38 of a sample across assay surface 10 and capture agent spots 20-28.Often, a sample passed through cartridge 100 is in liquid form, althoughthis is not essential. An analyte could, for example, be contained in agaseous fluid and passed through cartridge 100 in a manner similar topassage of a liquid. Similarly, as will be described in greater detailbelow, a fluid sample may typically be passed through cartridge 100under pressure through use of a pump or similar device. As such, a pumpand control valve may be connected to inlet 30 and/or outlet 34 suchthat a fluid sample is passed into inlet 30, across assay surface 10,and out outlet 34 by force (e.g., positive pressure exerted by a pumpconnected to inlet 30 and/or negative pressure exerted by a pumpconnected to outlet 34). For purposes of illustration and simplicityonly, embodiments of the invention will be described below in thecontext of analysis of a liquid sample.

FIG. 1B shows a schematic side view of cartridge 100 in use fordetection of an analyte according to an embodiment of the invention. Aliquid sample containing a labeled analyte 48 is being passed throughcartridge 100, forming analyte-capture agent complexes at capture agentspots 20-28. Labeled analyte 48 may be labeled using, for example, afluorescent label and/or a luminescent, colormetric, or radioactivelabel. Other known or later-developed labeling methods and techniquesmay similarly be used in practicing the various embodiments of theinvention. For purposes of illustration and explanation only, andwithout limitation to the scope of the invention, the embodiments of theinvention disclosed herein will be described in the context of analytesthat have been labeled with a fluorescent label.

An excitation beam 50 is positioned onto capture agent spot 22 to excitea fluorescently-labeled analyte. It will be understood by those skilledin the art that such an excitation beam could be a laser beam thatcovers a wide area of the array upon exposure, and not just one spot asused for illustrative purposes herein. As shown in FIG. 1B, excitationbeam 50 is delivered onto capture agent spot 22 from beneath. It isnoted, and would be recognized by one skilled in the art, of course,that excitation beam 50 may be delivered from other positions, includingfrom above and/or the side of capture agent spot 22.

In the embodiment shown in FIG. 1B, excitation occurs during the passageof the labeled analyte 48 through cartridge 100. As a consequence, bothfree analyte and analyte bound in an analyte-capture agent complex willbe excited and contribute to a fluorescent emission 52. That is,fluorescent emission 52 will contain fluorescence originating from boththe analyte-capture agent complex and free analyte, the latterconstituting background fluorescence that must be accounted for duringquantitative analysis. For the sake of simplicity, excitation of andfluorescent emission from only capture agent spot 22 is shown in FIG.1B. In some embodiments of the invention, each capture agent spot 20-28will be excited simultaneously or nearly simultaneously. In otherembodiments of the invention, excitation of and fluorescent emissionfrom a first capture agent spot or group of capture agent spots will betemporally separated from excitation of and fluorescent emission from asecond capture agent spot or group of capture agent spots. This may bedesirable, for example, where the first capture agent spot or group ofcapture agent spots targets analytes labeled with a first fluorescentlabel and the second capture agent spot or group of capture agent spotstargets analytes labeled with a second fluorescent label.

Similarly, as will be recognized by one skilled in the art, theexcitation and emission wavelengths will vary based on the fluorescentlabel(s) used. DYLIGHT® 549 (a fluorescent dye) and DYLIGHT® 647 (afluorescent dye), for example, commonly used as fluorescent labels, haveexcitation peaks at 549 nm and 647 nm, respectively.

A longitudinal assay method according to one embodiment of the inventioncomprises iteratively exciting one or more capture agent spot 20-28 andmeasuring the resulting fluorescence, as in FIG. 1B, to create a bindingcurve for the analyte. FIG. 2 shows a flow diagram of an illustrativemethod for creating such a binding curve according to one embodiment ofthe invention. An analyte may optionally be labeled at S1, in the casethat the analyte is not already labeled. As shown in FIG. 1B, theanalyte 48 may be fluorescently labeled, although this is not essential.Other labeling techniques or methods may be used, such as luminescence,colormetric, or radiolabeling.

At S2, a sample, such as a liquid, containing the labeled analyte 48 ispassed across an assay surface 10 containing a capture agent (e.g.,capture agent spots 20-28). At S3, the labeled analyte 48 is detected.In the case of a fluorescently labeled analyte 48, such detection may beby excitation and measurement of emission, as shown in FIG. 1B. S2 andS3 are iteratively looped at least once to obtain at least twomeasurements of emission intensity. In practice, S2 and S3 would likelybe iteratively looped at least twice to obtain at least threemeasurements of emission intensity. These measurements of emissionintensity (or other measure of intensity if a different labeling methodor technique is used) are used at S4 to create a binding curve for theanalyte. Binding curves created according to embodiments of theinvention, as well as their interpretation, will be described in greaterdetail below.

Returning to FIG. 1C, another embodiment of the invention is shown.Here, rather than exciting and measuring fluorescent emission (i.e.,detecting) while the labeled analyte (48 in FIG. 1B) is passing throughcartridge 100, excitation and emission occur during or following thesubsequent passage of a non-labeled solution 68, such as a buffersolution. That is, the labeled analyte 48 is passed through cartridge100 without detection, which occurs later, as shown in FIG. 1C, duringor following passage of the non-labeled solution 68. In such anembodiment, fluorescent emission 54 will be free or substantially freeof background fluorescence attributable to labeled analyte 48.

The method of FIG. 1C is shown in the flow diagram of FIG. 3. At S5, ananalyte may optionally be labeled, as described above. At S6, a samplecontaining the labeled analyte is passed across an assay surfacecontaining a capture agent. At S7, a non-labeled solution is passedacross the assay surface. At S8, the labeled analyte is detected (e.g.,by excitation and measurement of fluorescent emission). As noted above,detecting the labeled analyte at S8 may occur during and/or followingpassage of the non-labeled solution at S7. S6 through S8 are iterativelylooped to obtain at least two detections of the labeled analyte, whichare used at S9 to create a binding curve for the analyte. Similarly toas noted above, in practice, S6 through S8 would likely be iterativelylooped at least twice to obtain at least three detections of the labeledanalyte.

Longitudinal assays according to embodiments of the invention may becarried out in other ways, of course. For example, FIGS. 4A-4C show yetanother embodiment of the invention. In FIG. 4A, an unlabeled analyte 78is passed across assay surface 10, whereupon analyte-capture agentcomplexes are formed at capture agent spots 20-28. In FIG. 4B, afluorescently labeled solution 88 is then passed across assay surface10. In such an embodiment, the fluorescently labeled molecules in thesolution 88 bind to or otherwise complex with the analyte or theanalyte-capture agent complex, permitting development and quantitationof the analyte. As noted above, while the embodiment described hereutilizes a fluorescent label, this is not essential. Other labelingmethods and techniques may be similarly employed.

In FIG. 4C, a non-labeled solution, such as a buffer or, in someembodiments of the invention, sample solution containing the unlabeledanalyte 78, is again passed across assay surface 10, during and/orfollowing which detection (by excitation 50 and emission 54 measurement)occurs.

A flow diagram of the embodiment of FIGS. 4A-4C is shown in FIG. 5. AtS10, a fluid (e.g., liquid) sample containing the unlabeled analyte ispassed across the assay surface and capture agent. At S11, a solutioncontaining a label (e.g., fluorescent, luminescent, etc.) is passedacross the assay surface and capture agent. At S12, a non-labeledsolution, such as a buffer or a sample solution containing the unlabeledanalyte, is passed across the assay surface, during or following whichthe now-labeled analyte or analyte-capture agent complex is detected atS13. S11 through S13 are iteratively looped at least once to obtain atleast two detections at S13, from which a binding curve is created atS14. As noted above, S11 through S13 may, in practice, be looped atleast twice to obtain at least three detections at S13.

FIG. 6 shows a flow diagram of a method according to yet anotherembodiment of the invention. At S15, a sample containing an unlabeledanalyte is passed across the assay surface containing a capture agent.At S16, a labeled solution is passed across the assay surface. At S17,the analyte and/or analyte-capture agent complex is detected. In someembodiments of the invention, the detection at S17 occurs during passageof the labeled solution at S16, in which case the detected intensitywill include background fluorescence attributable to the label containedwithin the labeled solution, as in the embodiment shown in FIG. 1B. Suchbackground fluorescence may be accounted for during subsequentquantitative analysis.

At S18, a binding curve is created using the detection made at S17. AtS19, if an additional detection is needed (e.g., if only a singledetection at S17 has been made and the binding curve created at S18includes only the single detection), an additional sample containing theunlabeled analyte is passed across the assay surface at S20 and S16through S18 are then iteratively looped. If an additional detection isnot needed (e.g., if an additional sample containing the unlabeledanalyte has been passed across the assay surface at S20 at least once),the binding curve created during the most recent instance of S18 may beconsidered a final binding curve and flow may end.

While the methods above describe obtaining at least two detections of alabeled analyte or analyte-capture agent complex for use in creating abinding curve, as noted above, in practice, the number of detectionswill often be greater. For example, longitudinal assays according tovarious embodiments of the invention may be conducted over extendedobservation periods, typically between about 5 and about 120 minutes,with a detection made every minute or two.

In some embodiments of the invention, a detection may be made even morefrequently and the longitudinal assay may be conducted over time periodsof less than 5 minutes. For example, if the binding kinetics of aparticular assay permit detection every 10 seconds or so, enough datamay be collected to create a binding curve in less than a minute. Aswill be recognized by one skilled in the art, the binding kinetics ofassays vary considerably. As such, the durations of longitudinal assaysaccording to the various embodiments of the invention will depend, forexample, on the frequency at which detections may be made and the numberof detections desired for use in creating a binding curve.

As a consequence of the relatively fine grain of the resulting bindingcurves that may be created according embodiments of the invention, anadditional difficulty common to end-point assays, i.e., distinguishingspecific binding from non-specific binding, may be overcome, as will bedescribed below.

FIG. 7 shows a simplified binding curve for an analyte, as may becreated using a longitudinal assay method according to an embodiment ofthe invention (or other small or macro molecule screening platforms thatcan generate a temporal binding curve). As can be seen, the detectedintensity exhibits a steep initial rise early in the assay period,followed by a prolonged, substantially linear rise thereafter. The shapeof the detected intensity curve is due, in part, to the relativeproportion of analyte in a sample, and in part, to the specifics of theconcentration corrected binding interactions that are forming the curve.

With a specific binding event, a linear binding curve is observed thattypically does not reach equilibrium during the course of the assay (anassay may typically be run for 15 minutes, 30 minutes, 60 minutes, 120minutes, etc., and will depend on target analytes within the assayand/or the type of assay). The longer the assay is run, the higher thespecific signal builds, ultimately approaching the detection platformsmaximum intensity, depending on the concentration of the analyte.

In contrast, with a non-specific binding event, a binding curve isobserved that typically has a steeper initial gradient and reachesequilibrium (levels off) at an intensity level substantially below theplatform's maximum (in some cases at or under 1/20^(th) of platformsaturation). Furthermore, such non-specific signals rise to equilibriummore rapidly than the specific signals, and typically reach equilibriumduring the course of the assay (under 60 minutes, 45 minutes, 30minutes, etc., and often under 20 minutes or 10 minutes).

Whereas the intensity of the signal from a given binding event does notprovide a clear mechanism for distinguishing specific signals andnon-specific signals, as at any one time point the intensity level foreach may be similar, the behavior of the binding curve does provide amechanism for such discrimination. Indeed, if a signal from a bindingevent builds to an equilibrium level that is under 1/10^(th) of typicalplatform saturation levels within 40 minutes, or preferably within 30minutes, or preferably within 20 minutes, or preferably within 10minutes (depending on the specifics of the assay), then this signal maybe deemed to be the result of a non-specific binding event.

In contrast, if the signal from a given binding event builds throughoutthe assay in a linear or near linear way, and/or if the signal does notreach equilibrium within an assay period (e.g., within 30 minutes, 40minutes, 50 minutes, 60 minutes, 90 minutes, or 120 minutes, dependingon the assay parameters) the signal is deemed to be the result of aspecific binding event.

In the case where both specific and non-specific binding events form thesignal, a discrimination method according to an embodiment of theinvention can be used to isolate the non-specific component of thebinding curve form the specific component of the binding curve.Optionally, using curve fitting software to ‘fit’ appropriate rateequations to each curve and enable subsequent analysis (e.g., analysisof a combined curve will first see the non-specific componentsubtracted, then use the ‘fitted’ rate equation for the specificcomponent to back calculate initial concentration levels of the targetanalyte).

Some reasons for the observed differences in behavior of binding curvesresulting from either non-specific or specific interactions include, butare not necessarily limited to:

-   -   1. Concentration differences: For example, in a sample in which        the analyte of interest comprises less than 1% of the total        sample concentration, as is often the case, binding of the        analyte and capture agent and/or binding of the label and        analyte will increase gradually in a substantially linear        fashion, shown in FIG. 7 as the specific binding component. As        used herein, a “low abundance” analyte is any analyte having a        relative proportion of less than about 1/10 of the total sample        concentration. Often, the relative proportion of a low abundance        analyte is less than about 1/100. In some cases, particularly        where the low abundance analyte is a protein, the relative        proportion is less than about 1/1000 of the total sample        concentration.        -   Contrarily, in a sample in which non-analyte components            (e.g., non-analyte proteins, peptides, antibodies,            auto-antibodies, native antigens, protein complexes, lipids,            DNA, RNA, or other small or macro molecules, etc.) comprise            a much greater proportion of the sample, e.g., 10% or more            of the total sample concentration, as is often the case,            non-specific binding of a non-analyte component and capture            agent and/or binding of the label and non-analyte component            will increase to equilibrium very rapidly. This is shown in            FIG. 7 as the non-specific binding component.    -   2. Inherent Reaction Rate Differences: As used herein the        ‘inherent’ reaction rate is the rate of the reaction (binding        event) after being corrected for concentration differences.        Conversely, the reaction rate is the inherent rate multiplied by        the concentration of reactants. The ‘inherent’ reaction rate can        also be described as the rate constant in a mathematical        equation describing a reaction rate.        -   Typical capture agent-to-analyte bindings or reactions, e.g.            antibody to antigen binding, result through a mix of            complementarity in shapes (commonly known as ‘lock and key’            or ‘induced fit’ in the case of antibody to antigen            binding), hydrophobic interactions, hydrogen bonds,            electrostatic, and Van der Waals forces, and, in the cases            of some macro or small molecules, binding may result from            covalent interactions.        -   Importantly, the binding of a ‘non-specific’ interaction is            typically both ‘weaker’ and happens ‘faster’ than the            binding of a ‘specific’ interaction. More broadly, the            binding curve profile of a specific interaction typically            differs from that of a non-specific interaction in a given            assay. For example, for a specific antibody/antigen pair,            the primary component in the binding process is the ‘lock            and key’ or ‘induced fit’ interaction. Non-specific            interactions with either the antibody or antigen will not            include this ‘lock and key’ component. Instead, they will be            driven by, for example, Van der Walls forces. Such            interactions are typically weaker, occur faster, and            disassociate (end) faster, than the more specific ‘lock and            key’ interactions.        -   Accordingly, such reactions will typically build a signal            more rapidly, due to their faster initial binding rates            (typically represented by a higher rate constant and            ‘steeper’ initial binding curve), and the signal will also            reach equilibrium more rapidly, due to the weaker binding            from such reactions, and faster disassociation rates (the            rates of binding and disassociation level out after a            relatively short time period, e.g. 10 minutes, 20 minutes,            30 minutes, etc., depending on the specifics of the binding            kinetics).

In general terms, a higher rate constant is observed as a ‘steeper’binding curve on the plots in FIGS. 7-9. Non-specific binding eventsproduce steeper binding curves which reach equilibrium or saturationmore rapidly than will a ‘specific’ binding event. Specific bindingbuilds more gradually over time (i.e., specific binding has a lower rateconstant/gradient), and is represented in FIGS. 7-9 as the shallowercurves (lower rate constants), which typically continue to grow in alinear or near linear fashion over the course of the assay, asequilibrium for specific interactions may not be reached during the runtime of the assay.

In some cases, the signals detected from an assay will be a combinationof both specific binding and non-specific binding, shown as the solid“detected intensity” lines in FIGS. 7-9.

While it is known that a detected intensity will often include bothspecific and non-specific components, known end-point assaymethodologies preclude distinguishing these components. For example,using a known end-point assay, only a binding “snapshot” will beobtained, as shown in FIG. 7 at 60 minutes. Using such a methodology, asingle point (and therefore a single value) along the detectionintensity curve will be obtained. The specific binding and non-specificbinding components of that value are unknown and cannot be determinedusing known end-point assay methods.

Thus, when employing such a known method, one is left to assume that thetrue value of the specific binding component is less than the detectedintensity. Because one cannot know the extent of the non-specificbinding component, however, one cannot know how much lower the specificbinding value is from the detected intensity. Differences in sampleconstitution, the binding kinetics of various analytes, and othervariables can lead to large differences in the relative proportions ofthe non-specific binding components, making the assumptions required inusing known end-point assays highly unreliable.

For example, FIG. 8 shows a binding curve in which the non-specificcomponent is proportionally much greater than in FIG. 7. This may beattributable, for example, to the sample comprising a smaller proportionof analyte and a larger proportion of non-analyte components. In somecases, a signal will be the result of non-specific binding only, as noneof the target analyte is detectible. This may be because it is notpresent, or not present in high enough concentration to be detected, inwhich case the specific component of the combined curve will not bepresent. Alternatively, in some cases, the signal may arise fromspecific binding events only, in which case the non-specific componentof the combined curve will not be present.

As can be seen in the example of FIG. 8, a known end-point assay at 60minutes would result in a detected intensity approximately twice asgreat as the specific binding component. As discussed, in some cases,there may be no specific binding component to a signal, and the observedsignal is a result of non-specific binding only. End point or singletime point assays offer no mechanism for differentiating such signals.In a clinical setting, partial or total non-specific binding may lead tomisdiagnosis of a disease or disorder and/or suboptimal treatment ofsuch a disease or disorder. In a research or discovery setting, partialor total non-specific binding may lead to misidentification and/orpursuit of an analyte that is of little or no clinical, therapeutic, ordiagnostic value. It should be noted that the ‘binding curve’ analysismethodology described herein can be utilized with any platform thatcollects ‘time-course’ data, and thus enables the construction ofbinding curves and the quantification of associated rate constants andanalyte concentrations etc. Accordingly, the method is not exclusivelylimited to the longitudinal assay platform described herein.

FIG. 9 shows a detailed view of a portion of the binding curve of FIG.8. As can be seen in FIG. 9, at approximately point N in the detectedintensity curve, the rapid rise in signal intensity, attributableprimarily to non-specific binding, has begun to slow as the non-specificbinding approaches equilibrium. Thus, one can infer the equilibriumpoint of the non-specific binding at an approximate signal intensityN_(i) and elapsed time N_(t). Thus, one can determine or approximate thespecific binding component of the detected intensity by subtracting fromthe detected intensity value at any time greater than N_(t), the valueof N_(i).

Similarly, at approximately point S, a substantially linear rise in thedetected intensity curve begins. This substantially linear rise isprimarily or exclusively attributable to the specific binding component.Point S corresponds to a signal intensity S_(i) and elapsed time S_(t),which are greater than N_(i) and N_(t), respectively.

As will be apparent to one skilled in the art, known end-point assaysprovide insufficient data to differentiate signals caused by specificbinding from those caused by non-specific binding; neither can theydifferentiate the specific binding and non-specific binding componentsof the detected intensity where a signal contains components of each. Aneffective real-time longitudinal assay according to an embodiment of theinvention, however, permits such differentiation.

FIG. 10 shows a flow diagram of a method of differentiating specificbinding and non-specific binding using a longitudinal assay bindingcurve according to an embodiment of the invention. At S21, a pluralityof temporally-spaced detections of a capture agent to sample bindinginteractions are obtained, as described above, for example. As notedabove, such a plurality of detections may include as few as twodetections but may more typically include between about 5 and about 20detections. In some embodiments of the invention, such a plurality mayinclude more than about 100 detections.

At S22, it is determined whether the binding curve includes a rise toequilibrium at a first intensity by a first temporal point. If so (i.e.,Yes at S22), the first intensity value may be attributed to non-specificbinding at S23. As described above, the first intensity value may thenbe subtracted from intensity values at later temporal points todetermine or approximate a specific binding intensity value.

It should be noted, of course, as described above with respect to FIG.9, that the equilibrium at S22 is an inferred equilibrium. That is,referring again to FIG. 9 for purposes of illustration, the equilibriumis inferred to be at intensity N_(i), which is reached at temporal pointN_(t). In other words, in the case that the binding curve representsboth specific binding and non-specific binding, the non-specificequilibrium or saturation point is inferred from a change in gradient ora change in rate constant. This can be seen in FIG. 9 at N_(i) andN_(t), where the slope of the measured intensity decreases.

If a rise to equilibrium at a first intensity by a first temporal pointcannot be determined (i.e., No at S22), it may be determined at S24whether a linear or near linear rise begins at a second intensity by asecond temporal point. If so (i.e., Yes at S24), the linear or nearlinear rise from the second temporal point may be attributed to specificbinding, as described above.

As noted above, of course, it is possible that a binding curve will notrepresent any non-specific binding, i.e., all of the binding, or atleast all of the measured binding, is specific binding. In such a case,the linear or near linear rise determined at S24 may, in fact, compriseall of the binding curve and a change in gradient or rate constant willnot be observed.

On the other hand, it is also possible that a binding curve will notrepresent any specific binding, i.e., all of the binding, or at leastall of the measured binding, is non-specific. In such a case, as notedabove, it is likely that a true equilibrium will be reached within theassay period and no linear or near linear rise in measured intensitywill be observed.

It should be noted that following S21, either or both paths from S22 andS24 may be followed. That is, the determinations at S22 may be made inthe alternative, in combination, or sequentially. Thus, in the case thatflow passes initially from S21 to S24, if a linear rise at a secondintensity by a second temporal point cannot be determined (i.e., No atS24), flow may pass to S22.

Embodiments of the invention may be employed in any number of clinical,research, discovery, or developmental contexts. For example, indiscovery contexts, rapidly distinguishing specific and non-specificbinding in a detected intensity may be used in identifying andvalidating new biomarkers for therapeutic and/or diagnostic use. Suchrapid distinguishing may be used to dramatically enhance the discoveryprocess by only passing ‘real’ signals through to subsequent researchand bio-informatics analysis.

In a clinical context, embodiments of the invention may be used toscreen for known biomarkers as part of a clinical diagnostic test,whereby accurately quantifying true biomarker concentrations (i.e., thespecific binding component) enables more accurate and specificdiagnosis. In such contexts, embodiments of the invention may also lowercosts by lowering sample requirements.

As noted above, it may be beneficial in some cases to make adetermination of an initial concentration of an analyte. For example, inmost discovery contexts, analytes of interest will be of low abundance(i.e., less than about 1/10, more likely less than about 1/100, andstill more likely less than about 1/1000 of the total initial sampleconcentration).

One method of determining an initial concentration of an analyte, whichmay be employed in various embodiments of the invention, includesmeasuring a rate constant from a known concentration standard. This maybe done at the same time as or prior to the collection of specific andnon-specific binding data, using the same instrument parameters. Theknown data are fit to an equation to relate signal intensity toconcentration. An initial concentration of the analyte may then bedetermined by relating a concentration at a time point to an initialconcentration.

The quality of an initial concentration determined according to themethod above may be determined by examining the quality of the data fitbetween the known and unknown concentrations. In essence, this is ameasure of how well the real data fit the calculated data using the“best fit” parameters. With each increasing time point in a time courseexperiment, this measure can be recalculated to determine when anacceptable quality measure is met. Once met, data collection may cease.A minimum of three time course data points beyond time zero arenecessary, since fitting to two data point will always give a 100% fit.

Furthermore, where binding curves are made up of a specific andnon-specific component, two curves will need to be ‘fitted’ and two rateconstants calculated to facilitate the specific to non-specific bindingcalculations.

Other methods, including methods employing equations other than thosereferred to above, may be employed in calculating the rate constant andan initial concentration of an analyte, as will be known to one skilledin the art. Such other methods and equations are within the scope of theinvention, the above method being provided merely for the sake ofillustration.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the disclosure.As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any related or incorporated methods. Thepatentable scope of the invention is defined by the claims, and mayinclude other examples that occur to those skilled in the art. Suchother examples are intended to be within the scope of the claims if theyhave structural elements that do not differ from the literal language ofthe claims, or if they include equivalent structural elements withinsubstantial differences from the literal language of the claims.

What is claimed is:
 1. A method of detecting and determining aconcentration of a labeled analyte in a fluid sample, the methodcomprising: passing the fluid sample containing the labeled analyteacross at least one assay surface containing a capture agent for thelabeled analyte; detecting the labeled analyte and determining a firstdetection intensity at a first temporal point; repeating the passing anddetecting steps at least once using the same fluid sample anddetermining at least one additional detection intensity at at least oneadditional temporal point; creating a binding curve for the labeledanalyte including the first and at least one additional detectionintensities; and determining a concentration of the analyte in the fluidsample based on a comparison of the first detection intensity and the atleast one additional detection intensity in the binding curve.
 2. Themethod of claim 1, wherein the analyte and the capture agent are eachindependently selected from a group consisting of: a protein; a peptide;an antibody; an auto-antibody; an antigen; a native antigen; a proteincomplex comprising a complex of a protein and at least one of thefollowing: an antibody, an antigen, a native antigen, or anauto-antibody; a lipid; DNA; and RNA; and wherein the capture agent mayfurther comprise a cell or tissue lysate or fractions thereof.
 3. Themethod of claim 1, wherein the labeled analyte is labeled with at leastone of the following: a fluorescent marker, a luminescent marker, acolormetric marker, or a radioactive marker.
 4. The method of claim 1,wherein the fluid sample is a liquid.
 5. The method of claim 1, whereinthe detecting occurs during the passing of the fluid sample.
 6. Themethod of claim 1, wherein the detecting occurs after the passing of thefluid sample.
 7. The method of claim 1, wherein repeating the passingand detecting steps at least once includes repeating the passing anddetecting steps between about 5 times and about 20 times.
 8. The methodof claim 1, further comprising: distinguishing specific binding fromnon-specific binding in the detected labeled analyte using the bindingcurve and/or a constant associated with the binding curve.
 9. The methodof claim 8, wherein the distinguishing specific binding fromnon-specific binding comprises: determining a first detection intensityand a first temporal point at which a rise to an equilibrium may beinferred; and attributing to non-specific binding a portion of at leastone detected intensity subsequent to the first temporal point that isequal to the first detection intensity.
 10. The method of claim 9,wherein distinguishing specific binding from non-specific bindingfurther includes: subtracting from a second detection intensity thevalue of the first detection intensity; and attributing to specificbinding a remaining portion of the second detection intensity, whereinthe second detection intensity is greater than the first detectionintensity and the second detection intensity occurs at a second temporalpoint that is subsequent to the first temporal point.
 11. The method ofclaim 8, wherein distinguishing specific binding from non-specificbinding includes: determining a first detection intensity and a firsttemporal point at which a rise to an equilibrium has been reached; andattributing the first detection intensity to non-specific binding. 12.The method of claim 11, wherein distinguishing specific binding fromnon-specific binding further includes: attributing detection intensitiesoccurring subsequent to the first temporal point to non-specificbinding; and concluding that the detection intensities of the bindingcurve do not include a specific binding component.
 13. A method ofdetecting and determining a concentration of an analyte in a fluidsample, the method comprising: passing the fluid sample containing theanalyte across at least one assay surface containing a capture agent forthe analyte; passing a solution containing a labeled molecule across theat least one assay surface to label at least a portion of the totalanalyte in the fluid sample, the labeled molecule being capable ofbinding to the analyte; passing a non-labeled solution across the atleast one assay surface; detecting the labeled molecule and determininga first detection intensity at a first temporal point; repeating thepassing steps and the detecting at least once using the same fluidsample and determining at least one additional detection intensity at atleast one additional temporal point; creating a binding curve for theanalyte including the first and at least one additional detectionintensities; and determining a total concentration of the labeled andunlabeled analyte in the fluid sample based on a comparison of the firstdetection intensity and the at least one additional detection intensityin the binding curve of the labeled molecule.
 14. The method of claim 1,further comprising: passing a non-labeled solution across the at leastone assay surface.
 15. A method of detecting and determining aconcentration of an analyte in a fluid sample, the method comprising:passing the fluid sample containing the analyte across at least oneassay surface containing a capture agent for the analyte; passing asolution containing a labeled molecule across the at least one assaysurface, the labeled molecule being capable of binding to the analyte;detecting the labeled molecule and determining a first detectionintensity at a first temporal point; repeating each of the passing stepsand the detecting step at least once and determining at least oneadditional detection intensity at at least one additional temporalpoint; creating a binding curve for the analyte including the first andat least one additional detection intensities; and determining aconcentration of the analyte in the fluid sample based on a comparisonof the first detection intensity and the at least one additionaldetection intensity in the binding curve.
 16. The method of claim 15,further comprising: passing a non-labeled solution across the at leastone assay surface after passing the fluid sample across the at least oneassay surface.
 17. The method of claim 15, further comprising: passing anon-labeled solution across the at least one assay surface after passingthe solution containing the labeled molecule across the at least oneassay surface.